Analyte assays and devices

ABSTRACT

The present invention provides a method and a device that utilizes capillarity-mediated, chromatographic transport, for the qualitative or semi-quantitative analysis of selected analytes in liquid samples. The device utilizes an applicator/collection device for collecting and administering the sample to the flow path such that reagent(s) flow through the applicator/collection device, washing the sample into the reaction pathway. The device further utilizes an air gap between the initial location of the reagent and the reaction pathway to funnel the reagent efficiently through the sample so as to collect all or substantially all of the sample and make it available for the reaction(s).

This application is a continuation of application Ser. No. 09/105,822filed Jun. 26, 1998, now U.S. Pat. No. 6,235,539, which is acontinuation-in-part of application Ser. No. 08/736,801, filed Oct. 25,1996, now abandoned.

FIELD OF INVENTION

The present invention relates to improved analyte assays and devices forquantitative or qualitative assays of a member of any type of bindingpair such as ligand-receptor, antigen-antibody, etc., as well as forsubstrates or products or products of chemical and biochemicalreactions. This includes immunometric assays, which are designed for theanalysis of selected analyte(s) in a sample.

BACKGROUND OF INVENTION

Today binding-pair assays are utilized extensively in such fields asclinical, forensic, and veterinary medicine, pharmacological testing,environmental monitoring, food quality assurance, and other relatedareas. All these fields have needs for rapid and effective analysis ofspecific substances (referred to as analytes), which are frequentlyfound in low concentrations within the given test sample. The basicprinciples and mechanisms of assays are specifically designed toaccommodate such urgent needs.

These assays are especially useful in the in vitro determination of thepresence and concentration of analytes within physiological fluids. Forexample, the determination of specific proteins, enzymes, hormones,metabolites, and therapeutic or toxic drugs within the blood, urine, orcerebral spinal fluid has greatly enhanced the efficacy of diagnosticmethodologies in clinical medicine.

Moreover, the development of non-radioactive labeling components, whichallow the direct visualization of the completed reaction, hasfacilitated the use of binding-pair assay procedures outside of the“typical” laboratory. For example, in clinical office settings,non-radioactively-labeled, binding-pair assays are useful for providingrapid, simple procedures which may be performed while the patient isstill in the office. Thus diagnosis can be accomplished without delay,and treatment may be instituted during a single visit.

Without such assays, it was frequently necessary to collect the samplefrom the patient during a first visit and to have the biological sampleanalyzed by a clinical laboratory at a later time. During such time, thepatient was sent home and often required to return for a second officevisit in order to receive the proper treatment and/or medication. Suchdelay was at best inefficient, and at worst potentially lifethreatening.

The term “binding-pair assay” refers to an assay between twobinding-pair members designed to facilitate the formation of a complexbetween a particular analyte of interest and another substance capableof specific interaction with the analyte. In this way, the presence ofparticular analyte may be detected. Alternatively, the binding-pairmember may be a substance which, if detectable, may be used to infer thepresence or absence of the analyte within the sample.

In the context of the present invention, the term “analyte” refers to,but is not limited to, compounds such as proteins, modified proteins,peptides, nucleic acids such as deoxyribonucleic acid (DNA), ribonucleicacid (RNA), peptide nucleic acid (PNA), haptens, antigens, antibodies,and any metabolites of these substances and any other compounds, eithernatural or synthetic, which may be of diagnostic interest and which havea specific binding partner (e.g., the receptor moiety of aligand-receptor assay, or the substrate of an enzyme).

Binding-pair assays rely upon the binding of one analyte by its specificbinding partner to determine the concentration of the analyte within thetest sample. Binding-pair assays may be differentiated and categorizedas either competitive or non-competitive in nature. Non-competitiveassays generally utilize the receptor component in a substantial excessover the concentration of the analyte to be determined in the assay.

One type of non-competitive assay is usually referred to as “sandwichassay.” It employs the methodology whereby the analyte is detected viaits binding to two binding partners. One partner may be labeled tofacilitate a subsequent detection and the other is immobilized to asolid-phase to facilitate separation of the bound analytes from unboundreaction components (e.g., unbound labeled first receptor). An alternatenon-competitive assay can be termed a “blocking assay.” In this type ofassay, sample is first mixed with a binding partner (usually labeled),and any analyte in the sample binds to the binding partner. The mixtureis then allowed to react with analyte analog, which is usually bound toa solid phase. The more analyte is present in the sample, the morebinding sites on the binding partner will be blocked, and the less sitesthere will be for the analyte analog to bind. Thus, in this form ofassay, the more label is bound to the solid phase, the less analyte ispresent in the test sample.

In contrast, competitive binding-pair assays generally involve analytefrom the test sample, a purified binding partner or binding partneranalog that is labeled to facilitate detection, and a rate-limitingconcentration of binding-partner species. The sample analyte and thelabeled analyte/analyte analog moieties are subsequently allowed tocompete for the limited number of binding sites provided by the bindingpartner species present in the assay mixture.

Competitive binding-pair assays can be further differentiated as beinghomogeneous or heterogeneous in nature. In homogeneous assays, all ofthe reactants participating in the competition reaction are mixedtogether and the concentration of analyte is determined by its effect onthe extent of binding between its binding partner and labeled analyte.The signal observed is a direct function of this binding and can berelated to the overall concentration of analytes present in the testsample. U.S. Pat. No. 3,817,837, which is incorporated herein byreference, discloses a homogeneous, competitive immunometric assay inwhich the labeled analyte analog is a ligand-enzyme conjugate and thebinding partner is an antibody capable of binding either the analyte oranalyte analog. In general, homogeneous assay systems require both anexternal instrumentality to determine the result and the priorcalibration of the observed signal by separate tests performed withknown concentration of the specific analyte in a process known asstandardization. While homogeneous assays have dominated competitiveimmunometric assay system development, such systems are not capable ofproviding results for the determination of multiple analytes in a testsample in a single-test format not requiring external instrumentality.

Heterogeneous, competitive binding-pair assays require separation of thebound, labeled analyte or its binding partner from the free, labeledanalyte or its binding partner and a subsequent measurement of eitherthe concentration of the bound or free fraction. Methodologies forperforming these assays are described in U.S. Pat. Nos. 3,654,090,4,298,685, and 4,506,009, which are incorporated herein by reference.The quantitative or semi-quantitative measurement of analyteconcentration utilizing this methodology cannot be performed without theuse of additional tests to calibrate the assay results. Hence, only thepresence or absence of the analyte can be determined without additionalinstrumentation or tests. Recently, however, methods have been developedfor the internal calibration of binding-pair assays by providing adevice which incorporates reference zones whereby the given response atthe reference zone represents the assay response for a specificconcentration of the analyte. The response generated by the unknownconcentration of the analyte in the test sample is then compared withthe response at the reference zone to determine the concentration of theanalyte in the test sample in a qualitative or quantitative manner. U.S.Pat. No. 4,540,659, which is incorporated herein by reference, describesthe system that incorporates several analyte concentration standards.They provide the ability to make semi-quantitative determinations ofanalyte concentrations in competitive binding-pair assays through adirect visual examination.

Sample collection and application means are known within the relevantfield. For example, an applicator component, consisting of a separatewand-like component with a bibulous material attached to one end, isdescribed in U.S. Pat. Nos. 5,169,789 and 4,770,853, which areincorporated herein by reference. The sample is collected for assay bysimple absorption of the aqueous sample and subsequent placement of thecollection component into the assay device. U.S. Pat. No. 4,624,929,incorporated herein by reference, discloses a sample collector comprisedof a bibulous membrane confined in a housing. Collection of the sampleis facilitated by contacting the sample collector with the desiredaqueous sample. European Patent Application Nos. 88303744.2 and90301697.0, incorporated herein by reference, disclose a wick-likesample collector, comprised of bibulous material, which is contiguouswith the internal chromatography material.

While there are numerous assay devices and sample collection andapplication means that are currently in use within the relevant fields,the device disclosed herein serves to mitigate several of thedifficulties which are frequently encountered in the utilization ofthese devices. For example, the requirement for large initial samplevolumes, sometimes as much as several milliliters, often becomesproblematic with many commercially available devices. This volumerequirement is a function of the comparatively inefficient samplecollection and/or application means these devices possess. Moreover, therequirement for such large initial sample volumes can potentially leadto difficulties when only small sample volumes or only a single testsample is available.

In contrast, due to the utilization of a novel sample collector andapplicator, the present invention requires comparatively small initialsample volumes for analysis of analyte concentration. The features ofthe present invention also negate the need for secondary, externalsample collector/applicator, which many, if not all, of the currentlyutilized devices require. Furthermore, the present invention greatlyreduces the probability of sample contamination or cross-contamination,which is frequently encountered with the use of such secondary means. Anadditional unique feature of the sample collector/applicator componentof the present device is the ability to collect and solubilize a driedsample, which putatively contains the analyte, without the use ofsecondary instrumentalities or methodologies.

SUMMARY OF THE INVENTION

The present invention is directed to any type of binding-pair assay anddevice that utilizes chromatographic, capillarity-mediated transport forthe qualitative or quantitative analysis of selected analytes insamples. The disclosed assay system is comprised of a chromatographydevice which incorporates a sample collection and application componentfor the delivery of the solubilized sample directly into thechromatographic flow. The disclosed device fulfills numerous, unmetneeds within the relevant fields. It provides, but is not limited to,the following benefits:

-   -   (1) All required components of the assay system are contained        within a single unitized device that is capable of being        disassembled into a sample collector/applicator and detection        means;    -   (2) the assay system allows the collection of the sample and its        subsequent application to the chromatographic components in such        a way as to minimize any potential sample diminution due to        incomplete sample delivery;    -   (3) the assay system allows collection of a comparatively small        sample volume;    -   (4) the assay system negates the requirement of an additional        external instrumentality which collects and applies the sample        to the assay device;    -   (5) the assay system minimizes the potential for sample        contamination and/or cross-contamination caused by the use of        non-integrated, external sample collection and/or application        devices; and    -   (6) one embodiment of the present assay system discloses the use        of self-contained, breakable, reagent containers for the        delivery of various solutions utilized in the analysis of the        sample.

Currently, a major problem associated with binding-pair assays is thecollection of the test sample and its subsequent application to theassay device. Many, if not all, of the commercially available devicesrequire comparatively large sample volumes to be collected because thedevices are inefficient in delivering the applied sample to thechromatographic means. The present device, however, greatly mitigatesthese difficulties via a novel sample applicator, which utilizes anabsorbent wick-like component and air gap to facilitate sample deliveryto the associated chromatographic components contained within thedevice. Therefore, the sample putatively containing the analyte ofinterest can be transferred to the chromatographic means by having thereagent(s) flow through the sample collector/applicator, concomitantlywashing the sample onto the chromatographic means.

DESCRIPTION OF THE DRAWINGS

Those individuals skilled in the relevant art may better understand thepresent invention and appreciate its advantages by referring to theaccompanying drawings wherein:

FIG. 1 shows a side view of the device detailing: the sampler member,the chromatography components, and the housing for said device.

FIG. 2 shows a top view of the device detailing: the sampler member, thechromatography components, and the housing for said device.

FIG. 3 shows an orthographic view detailing the sampler member, thechromatography components, and an “exploded” illustration of the housingfor the device.

FIG. 4 shows an orthographic view detailing the reagent containers.There may be one or more of these containers associated with the device,depending upon the nature of the assay being performed.

FIG. 5 shows an orthographic view with the additional detection port.

FIG. 6 shows the sampler member with an optional “sharp” for drawingblood or other body fluids.

FIG. 7 shows the sampler with a sharp surrounding the absorbent wick ortubing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides binding-pair assays and devices thatutilize capillarity-mediated, chromatographic transport, for thequalitative or quantitative analysis of selected analytes in samples.The invention is useful for a wide variety of assays, both ligand-basedand non-ligand-based. Applicable ligand-based methodologies may include,but are not limited to, competitive immunoassays, non-competitive orso-called sandwich technique immunoassays, and blocking assays.

The use of the invention is not limited to immune systems. Other ligandassays include ligand-receptor assays, such as detecting insulin with aninsulin receptor, and ligand-binding protein type assays, such asdetecting certain antibiotics using a beta-lactam binding protein.Enzymes and their substrates can be determined as well. Applicablenon-ligand-based assay methodologies may include any type of chemicaland biochemical reactions that can be detected by a device that utilizeschromatographic, capillarity-mediated transport.

The present device comprises a housing which includes means for thecollection of a sample putatively containing the analyte of interest,its application into the assay device and means for the detection, suchas by direct visualization of the results of the binding-pair assay. Thedevice may also further comprise

-   -   (1) a housing with means enclosed therein for the conjugation of        a label to a specific analyte to be detected within the sample        (label transfer pad);    -   (2) a means for the sequestering the specific analyte within a        defined spatial location (capture zone); and    -   (3) a means for allowing liquids and any non-sequestered,        solubilized materials contained therein, to undergo        capillarity-mediated transport away from the capture location        for subsequent absorption in a discrete area (blotter pad).

The present invention has broad application. It may also be employed inany number of assays which utilize bibulous material to mediate the flowaway from an initial spatial location where the bibulous material iscontacted with a medium putatively containing either the analyte to bedetermined or reagents used for the analysis of the analyte.

Prior to proceeding with the detailed description of the specificembodiments of the present invention, a number of terms will be defined.

Definitions

Analyte: the substance or composition to be measured in the assay.

Analyte analog: a specific derivative of the target analyte which may beoptionally attached, either covalently or non-covalently, to otherchemical species (e.g., a label). The analyte analog may be used, forexample, to compete with the analogous target analyte for binding to thespecific binding partner (i.e., competition assay). Where themodification of the analyte provides means to join the analyte analog toanother molecule, or where the analyte has a functionality which is usedto bind directly to another molecule, the analyte portion of theconjugate will be referred to as an analyte analog.

Binding partner: a molecule, such as a receptor, binding protein,antibody or antibody fragment, or enzyme (which binds to its substrate),which possesses the ability to interact with another molecule in ahighly specific polar and spatial manner.

Antibody: an immunoglobulin, or a derivative or fragment thereof, whichis capable of specifically binding to an antigen in a receptor-ligandbased reaction. The antibody or fragment may be polyclonal ormonoclonal, or native or chimeric. The preparation of antibodies is wellknown in the art. For example, antibodies may be generated by theimmunization of a host followed by the collection of sera (polyclonal),or by hybridoma cell line-based technology (monoclonal).

Label: any molecule which is bound (via covalent or non-covalent means,alone or encapsulated) to another molecule or solid support and which ischosen for specific characteristics which allow detection of the labeledmolecule. Generally, labels are comprised of, but are not limited to,the following types: particulate metal and metal-derivatives,radioisotopes, catalytic- or enzyme-based reactants, chromogenicsubstrates and chromophores, fluorescent and chemiluminescent molecules,and phosphors. The utilization of a label produces a signal which may bedetected by means such as detection of electromagnetic radiation ordirect visualization, and which can optionally be measured.

Bibulous material: a porous material that is susceptible to traversal bya liquid medium in response to capillary force. Such materials can behydrophilic, or are capable of being rendered hydrophilic, and includenatural polymeric substances (e.g., cellulosic materials),fiber-containing papers (e.g., filter and chromatographic papers), andsynthetic or modified naturally-occurring polymers (e.g.,nitrocellulose, cellulose acetate, polyacrylamide, cross-linkeddextrose, or agarose), which are either utilized by themselves or incombination with other materials. The bibulous material may bepoly-functional, or be capable for being made poly-functional, forexample to permit covalent bonding of receptors, antibodies, or othercompounds which function as components of the specific assaymethodology.

Capillary Communicating Contact: When two elements of the device are incapillary communicating contact, the elements of the device are capableof transferring fluid from one element to the other, when fluid ispresent, by capillary action.

The Device

The present invention provides a methodology and device for any types ofbinding-pair assays, utilizing capillarity-mediated, chromatographictransport for the qualitative or quantitative analysis of a selectedanalyte putatively contained within a test sample.

FIGS. 1-6 depict a device for the detection of a specific analyte in asample comprising two distinct components: the Sampler (1) and theDetection (2) members. The Sampler member (1) is comprised of anabsorbent wick or tubing (6) which exhibits capillarity, and a reagentdelivery system (22). The wick or tubing (6) is enclosed in a housing(4) fabricated from a rigid or semi-rigid, non-water-permeable material.When a wick is used for element 6, it is comprised of a bibulousmaterial. When tubing is used for element 6, it may be capillary tubingof glass, plastic, or other appropriate material.

The reagent delivery system (22) can be contained within the housing(4), partially within and partially outside the housing (4), or fullyoutside the housing (4). Optionally, an absorbent reagent pad (3) isenclosed within the housing (4) as a part of the reagent delivery system(22). An opening, the reagent application port (5), can be optionallyprovided in the housing (4) of the Sampler member to facilitate theaddition of various solutions through the reagent delivery system (22),optionally through the absorbent reagent pad (3).

The wick or tubing (6) is in capillary communicating contact with thereagent delivery system (22) via the absorbent reagent pad (3) when itis present, or via any other source of reagent. A line of demarcation(7) can be utilized on the absorbent wick or tubing (6) to facilitatethe collection of a pre-determined volume of a liquid sample viacapillary action.

The Detection member (2) is comprised of a housing, fabricated from arigid or semi-rigid, non-liquid-permeable material, optionally providingan upper (8) and lower (9) section. The housing can serve to bothcontain and position various components of the assay device.

Contained within the housing is the chromatography region (24),utilizing a chromatography medium (10) which is comprised of at least atransit zone (20) and a capture zone (13). The transit zone (20) canoptionally include a proximally located label transfer pad (11). Whenutilized, the label transfer pad (11) contains a labeled, specific,analyte-binding reagent (L-SABR) or the components and means forassembling an L-SABR (for a non-competitive reaction) or a labeled,specific, analyte analog (L-SAA) or the components and means forassembling an L-SAA (for a competitive reaction). If components andmeans for assembling the L-SABR or L-SAA are used, the components mustbe capable of assembling and interacting in the form of an L-SABR withthe analyte, or in the form of an L-SAA with the binding partner. TheL-SABR or L-SABR, or its components, is retained within the labeltransfer pad (11) while the absorbent material comprising the pad is inthe dry state. When components and means for assembling an L-SABR orL-SAA are utilized, L-SABR or L-SAA is assembled prior to or whenanalyte moves through the transfer pad (11) during the course of theassay. The L-SABR or L-SAA (either initially present or subsequentlyassembled) becomes freely mobile through both the label transfer pad(11) (when used) and the porous chromatography medium (10) whencontacted by the assay liquid.

Alternatively or additionally, label and/or components and means forassembling the L-SABR or L-SAA can be added through a detection port(19) which enables detection in the Detection member (2) optionallylocated at the proximal end of the transit zone (20). The L-SABR orL-SAA thus added or assembled enters the transit zone and becomes freelymobile through both the label transfer pad (11) (when used) and theporous chromatography medium (10) when contacted by a solution.

The detection port (19) can also be used for the addition of buffer(s),reagent(s) and/or other reaction components to be used in the assay. Forexample, a reagent useful in visualizing the L-SABR or L-SAA can beadded through the detection port (19); likewise, a buffer forsolubilizing L-SABR or L-SAA entrapped in the label transfer pad (11)can be added through the detection port (19).

The chromatography medium (10) can optionally be in capillarycommunicating contact with a distally located blotter pad (12). Thechromatography medium (10) comprises a spatially-distinct locationdefined as the capture zone (13), said zone being located downstreamfrom the transit zone (20) and the optional label transfer pad (11)and/or the optional detection port (19) and upstream from the blotterpad (12). A non-reagent-soluble, non-labeled specific analyte-bindingreagent (SABR), or a specific analyte analog (SAA) is immobilized on thelabel transfer pad (11) when it is used. The L-SABR and the SABR canhave the same or different reactive sites, depending on the type ofassay to be performed. Likewise, the L-SAA and the SAA can have the sameor different reactive sites.

In one possible embodiment, the assay of the sample putativelycontaining the analyte of interest is initiated by a separation of theSampler (1) and Detection (2) members from one another. The distal endportion (16) of the absorbent wick or tubing (6) is next brought intocontact with the chosen liquid sample (e.g., a blood droplet). Thesample is drawn into the absorbent wick or tubing (6), via capillaryaction, until such time as the desired amount of sample has beencollected (for example, until the level of the collected liquid samplehas reached the line of demarcation (7)).

Optionally, a mechanism capable of puncturing skin to draw blood orother fluids (a “sharp”) (23) can be incorporated in the device.Preferably this sharp is retractable or can be easily removed from thesampler member (1) after puncture. FIG. 6 shows two versions of aretractable sharp. In another embodiment, illustrated in FIG. 7, thesharp can itself be concave or hollow and can surround the wick ortubing (6). The sample can then flow along the sharp body and be exposedto the distal end of the wick or tubing (6) to allow collection of thesample by the wick or tubing (6). Alternatively, the concave or hollowsharp can be positioned next to the wick or tubing (6) such that thesample will flow along the sharp body and then encounter the proximalend of the wick or tubing (6) at the base of the sharp, such as througha hole in the sharp. The sample would then proceed to flow through thewick or tubing (6).

The assay device is then reassembled by the insertion of the Samplermember (1) into the Detection member (2), such that the absorbent wickor tubing (6) is placed in capillary communicating contact with thechromatography medium (10) present at the proximal end of the labeltransfer pad (11). A solution (e.g., buffer) is then added to thereagent application port (5) such that the absorbent reagent pad (3)becomes saturated. The solution is drawn into the absorbent wick ortubing (6) by its inherent capillarity, causing the movement of the testsample through the absorbent wick or tubing (6) and into the labeltransfer pad (11).

An air gap (14) is utilized to separate the absorbent reagent pad (3)from the label transfer pad (11), with the air gap (14) being “bridged”by the absorbent wick or tubing (6) upon reassembly of the assay device.The utilization of the air gap (14) maximizes the transfer of the liquidsample contained within the absorbent wick or tubing (6) to the labeltransfer pad (11). It forces capillarity-mediated transfer of the liquidfrom the absorbent reagent pad (3) through the absorbent wick or tubing(6), concomitantly facilitating sample transfer.

In this embodiment, the movement of the test sample and liquid reagentinto the label transfer pad (11) causes the L-SABR or L-SAA to besolubilized, thus facilitating the formation of a complex between thislabeled moiety and any specific analyte putatively contained within thetest sample. When L-SABR is used, the analyte/L-SABR complexsubsequently interacts with, and is sequestered by, the immobilized,non-labeled SABR located within the capture zone (13). Depending uponthe type of label chosen, detection of this sequestered complex may beaccomplished by direct visualization via the transparent viewing port(15) located within the upper section (8) of the Detection member (2)over capture zone (13) to allow visualization of the capture zone (13).

Any dissolved, non-complex and non-sequestered materials found withinthe capture zone can be minimized by a continuous, capillary-based flowof the solution (e.g., buffer) applied to the absorbent reagent pad (3)which washes these non-sequestered components past the capture zone(13). Generally, this flow will continue until either the absorbentreagent pad is depleted or the blotter pad (12) becomes saturated.

In another embodiment, illustrated in FIG. 4, the device incorporatesone or more self-contained, breakable reagent container(s) (17). Thereagent container (17) encloses a solution which, when released, flowsinto and/or through the absorbent reagent pad (3). This solution mayoptionally contain a variety of substances including, but not limitedto, analyte, analyte analog, specific analyte -binding reagent, signalgenerating reagent (e.g., a substrate for an enzymatic label), or anyother ancillary reagent. The container may be integral with the housingof the device, separate therefrom, or both, where more than a singleself-contained reagent container is employed.

The device may optionally comprise a reagent application port (5), sothat at least one of the breakable containers may be positioned so as toallow fluid flow through the reagent application port (5) upon ruptureof the container(s) (17). Upon rupture, the enclosed solution isrendered capable of undergoing capillarity-mediated transport throughthe optional absorbent reagent pad (3) and the absorbent wick or tubing(6).

The reagent containers (17) themselves are preferably water-impermeablebefore breakage, and may be rigid, semi-rigid, or flexible. The solutioncontained therein is capable of being delivered to the assay device bycrushing, cutting, puncturing, melting, or otherwise rupturing thecontainer (17) or a seal between the container and the assay device.Materials utilized in the fabrication of the reagent containers mayinclude, but are not limited to, glass, polymers, fiber-reinforcedpapers, plastics, waxes, and other materials which meet the previouslydiscussed structural criteria. The container shape may be any shapewhich is spatially and sterically compatible with the present device(e.g., spherical, rectangular, ellipsoidal, and so-forth). The totalvolume of the reagent container (17) will vary, depending upon thespecific liquid reagent contained therein, the function of the givenreagent in the assay, the overall size of the assay device, the totalabsorptive capacity of the bibulous materials utilized in the assaydevice, the total number of reagent containers employed, and similarlimitations.

The housing comprising either or both the Sampler (1) and Detection (2)components of the assay device may be fabricated from variousmoisture-impermeable materials including, but not limited to, thermo-and vacuum-formed plastics, fiber-reinforced paper products, polymersand other appropriate materials. The material utilized to fabricate thehousing preferably does not interfere with the sample, the samplemedium, or any reagents used in the assay procedure. While a transparentmaterial can be used in the fabrication of the viewing port (15),alternative embodiments include no covering on the viewing port (15), ora large portion of the upper section (8) of the Detection component (2)being fabricated from transparent material.

Following the insertion of the various chromatographic materials duringthe construction of the device, the upper (8) and lower (9) sections ofthe Detection component (2) may be securely fastened together byultrasonic welding, adhesives, and other relevant methodologies.Alternately, the Sampler (1) and/or the Detection (2) components mayeach be fabricated in a single, unitized piece, which is subsequentlyused to enclose the various internal components.

Both the absorbent reagent pad (3) and blotter pad (12) may beconstructed from any bibulous, porous, or fibrous material that iscapable of absorbing a liquid. Examples include porous plastic polymermaterials including, but not limited to, polypropylene, high molecularweight polyethylene, polyvinylidene fluoride, acrylonitrile, andpolyterafluoroethylene; cellulosic materials (e.g., nitrocellulose); orheavy-weight, high-absorbency chromatographic paper. The use of achromatographic paper in the fabrication of the blotter pad (12) isgenerally preferable, due to its high degree of absorbency and low cost.Additionally, with respect to the absorbent reagent pad (3), it ispreferred that the chosen material retains some degree of structuralintegrity when saturated. Regardless of the material chosen, it may beadvantageous to pre-treat the member with a surface-active agent duringfabrication so as to reduce any inherent hydrophobicity andconcomitantly increase its ability to absorb and deliver liquid samplesin an efficacious manner.

It should also be noted that the proximal end (16) of the absorbent wickor tubing (6) member may be saturated with an inert dye (e.g., BlueDextran) to facilitate the visualization of the chromatographic flow ofcolorless solutions.

Chromatographic media (10) which may be utilized with the present deviceinclude those chromatographic substrate materials possessing capillarityand the capacity for chromatographic solvent transport ofnon-immobilized, liquid-soluble reagents and sample components. While awide variety of chromatographic materials used for paper chromatographyare suitable for use with this invention, the use of microporous ormicrogranular thin-layer chromatographic substrates is generallypreferred due to the marked increase in speed and resolution which thesematerials provide.

The chromatographic material is preferably inert, as well as physicallyand chemically non-reactive with any of the sample components, reagents,or reaction products. Preferably, microporous nitrocellulose materialmay be utilized with a high degree of efficacy in the disclosed device.Nitrocellulose has the added advantage that the specific analyte-bindingreagent (e.g., antibody) present in the capture zone may be immobilizedwithout prior chemical treatment.

In contrast, if the chromatography material is comprised of achromatographic paper, for example, the immobilization of the specificanalyte-binding reagent can be performed via chemical couplingmethodologies. Commonly used reagents include, but are not limited to,cyanogen bromide (CnBr), carbonyldiimididazole, or tresyl chloride.Additionally, reagents capable of blocking non-specific binding sites onthe chromatographic medium which might hinder chromatographic solventtransport of the liquid sample or reagents may also be employed in thefabrication of the present device. Commonly used reagents include, butare not limited to, bovine serum albumin (BSA), gelatin, and casein,which are preferably selected for their ability to not interfere with,or cross-react with, the sample components, reagents, or reactionproducts.

SABR which may be utilized with the present invention are readilyidentifiable to one of skill in the relevant art and include thosematerials which are members of a “specific binding pair” (e.g., ligandand receptor, antigen and antibody, or enzyme and substrate). Forexample, an analyte and a receptor are related in that the receptorspecifically binds to the analyte and possesses the capacity todifferentiate the analyte from other materials having similarcharacteristics. The methodologies and devices disclosed by the presentinvention are particularly useful in the practice of immunological assaytechniques wherein the specific analyte-binding reagents consist ofreceptors, antibodies, antibody fragments, or synthetic antibodies orantigens. The present device preferably employs a specific binding pairconsisting of the desired analyte and an antibody which is specific for,i.e., has a high affinity for binding to, said analyte.

The “label” utilized on the L-SABR or L-SAA disclosed in the presentinvention may be selected from, but is not limited to, the followingcategories: chromogens/fluorescers, particulate metals or theirderivatives, components of catalyzed or enzymatic reactions,chemiluminescent compounds, and radioactive isotopic labels.

Chromogens include those compounds which absorb light in a distinctiverange such that a specific color may be visibly observed (i.e., dyes),or emit light when irradiated with electromagnetic radiation of aspecific wavelength or wavelength range (i.e., fluorescent compounds).Illustrative dye types include, but are not limited to, quinoline,acridine, alizarin, cyanine and anthraquinoid dyes. Fluorescent compoundfunctional groups include, but are not limited to, porphyrins,2-aminoaphthalene, p,p′-diaminobenzophenone imines, 1,2-benzophenazin,and quaternary phenanthridine salts. By irradiating a fluorescer withlight of a specific wavelength, one may obtain a plurality of emissions,thus providing multiple measurable events.

Particulate metal sol-based label can be obtained by the direct orindirect coupling of the desired reaction component (e.g., antibody)with particles of an aqueous dispersion of a metal, metal compound, orpolymer nuclei coated with a metal or metal compound, having a particlesize of at-least 5 nm. The term “coupling” is understood within therelevant art to encompass any type of chemical or physical binding, andincludes covalent and hydrogen bonds, polar attraction, absorption, andadsorption. Metals utilized in this labeling methodology may include,but are not limited to gold, platinum, silver, copper, and iron. A metalsol is generally defined as a suspension of metal or metal-derivativeparticles which, due to their extremely small diameter, remain in asuspension solely because of the Brownian motion effects. Theparticulate metal sols to be used as labels may be prepared in a numberof ways which are in themselves known. For example, the preparation of agold sol has been described (G. Frens, 241 Natural Physical Science 20(1973)), which is incorporated herein by reference.

Catalyzed reactions may be either enzyme or non-enzyme based.Considerations which must be taken into account when using anenzyme-based label include, but are not limited to, enzyme stability,turnover rate, sensitivity of the reaction to environmental factors, thenature of the substrate and products, and the effect of conjugation onthe enzyme's catalytic properties. The methodologies involved inenzymatic labeling are well known within the relevant art. A preferredexample is horseradish peroxidase (HRPO), used in conjunction with1.7-dihydroxynaphthalene, and 4-amino-antipyrine hydrochloride.

An alternative form of labeling is the use of chemiluminescentcompounds. The chemiluminescent source comprises a compound that becomeselectronically excited following a chemical reaction and subsequentlymay either emit light in the visible range or donate energy to asecondary fluorescent acceptor compound. These include, but are notlimited to, the 2.3-dihydro-1,4-phthaiazinedione family (luminol), the2,4,5-triphenylimidazole family (lophine), and the para-dimethylaminooxalate-ester family (luciferase).

Radioisotopic labels are widely utilized in immunometric assays. Theradiolabel may be selected from, but is not limited to, the followingradioactive isotopes: ³H, ¹⁴C, ³²P, ¹²⁵I and ¹³¹I. Methods of labelingsubstances with radioactive labels are well known within the relevantart.

In the present invention, the utilization of metal sol-based labels ispreferred. For example, a gold sol label conjugated to the specificanalyte-binding reagent (e.g., antibody) may be utilized. This labelallows the end-point of the reaction to be visualized without the needfor any additional instrumentality.

The present invention clearly fulfills several unfulfilled needs withinthe field of binding-pair assays. The novel construction of the unitizedSampler member facilitates both the initial collection, and subsequentassay of the sample putatively containing the analyte. The utilizationof a novel absorbent wick/air gap feature in the Sampler component notonly minimizes the potential for the contamination andcross-contamination of the sample by not requiring a secondary externalmeans for sample collection (e.g., via syringe, pipette, capillary tube,etc.), but also maximizes the transfer of the sample to the assaydevice, thus minimizing the required sample volume.

The following specific embodiments, by-way of example, are descriptionsof the utilization of the disclosed invention for the purpose ofimmunometric determination of a specific analyte present in a sample.These examples are not meant to limit the scope of the present inventionin any manner, but instead, merely serve to illustrate some of manypossible embodiments.

EXAMPLE 1

With reference to FIGS. 1-3, the assay is begun by a separation of theSampler (1) and Detection (2) components from one another. The distalend (16) of the absorbent wick (6) is next brought into contact with thesample, a blood droplet. The sample is then drawn into the absorbentwick (6) via capillarity-mediated transfer until such time as the fluidlevel has reached the line of demarcation (7), thus providing a meansfor the collection of a pre-determined sample volume. The assay deviceis then reassembled by the insertion of the Sampler component (1) intothe Detection component (2), such that the absorbent wick (6) is placedin capillary communicating contact with the label transfer pad (11). Asolution is then added to the reagent application port (5) such that theabsorbent reagent pad (3) is saturated. The solution is subsequentlydrawn into the absorbent wick (6) by its inherent capillarity, thuscausing the movement of the sample through the absorbent wick (6) andinto the label transfer pad (11). The label transfer pad (11) contains aparticulate metal label which is conjugated to an antibody, with theantibody being specific for the analyte putatively contained within thetest sample. The particulate metal label/antibody conjugate (“labeledantibody”) is found in a dried, non-immobilized form within the labeltransfer pad (11). An air gap (14) is utilized to separate the absorbentreagent pad (3) from the label transfer pad (11), and is bridged by theabsorbent wick (6) upon reassembly of the assay device. The concomitantmovement of the test sample and solution into the label transfer pad(11) causes the labeled antibody to be solubilized, facilitating theformation of a complex between the labeled antibody and any specificanalyte putatively contained within the test sample. The analyte/labeledantibody complex subsequently interacts with, and is sequestered by, theimmobilized, non-labeled antibody located within the capture zone (13).Detection of this sequestered complex is visualized via a transparentviewing port (15) located within the upper section (8) of the Detectioncomponent (2) and over the capture zone (13). Any liquid soluble,non-reacting materials found within the capture zone (13) are minimizedby a continuous, capillary-based flow of the solution initially appliedto the absorbent reagent pad (3). This flow continues until either theabsorbent reagent pad (3) is depleted of solution or the blotter pad(12) becomes saturated.

EXAMPLE 2

With reference to FIGS. 1-3, the device is utilized as in Example 1 withthe exception that the labeled antibody has been localized to the distalend-portion (16) of the absorbent wick (6) rather than to the labeltransfer pad (11). This specific embodiment allows for thepre-incubation of the sample with the labeled antibody prior to both thereassembly of the Sample (1) and Detection (2) components and thesubsequent addition of a solution (e.g., buffer) to the reagentapplication port (5). This specific embodiment ensures that the labeledantibody reaches the test sample, by the process of diffusion, ingradually decreasing concentrations, due to the solubilizing of thelabeled antibody following the addition of a solution (e.g., buffer) tothe reagent application port (5). The timing between the initialcollection of the test sample and the subsequent reassembly/solutionaddition may be varied to allow for the adjustment of assay sensitivity.

EXAMPLE 3

With reference to FIGS. 1-3, the device is utilized as in Example 1 withthe exception that the labeled antibody has been localized to theproximal end-portion (18) of the absorbent wick (6) rather than to thelabel transfer pad (11). This specific embodiment ensures that thelabeled antibody reaches the test sample, by the process of diffusion,in gradually increasing concentrations, as opposed to the previousexample (Example 2) where the labeled antibody reaches the test samplein gradually decreasing concentrations, due to the solubilizing of thelabeled antibody following the addition of a solution (e.g., buffer) tothe reagent application port (5). Requirements for differing reactionkinetics may favor one embodiment over the other.

EXAMPLE 4

With reference to FIGS. 1-3, the device is utilized as in Example 1 withthe additional step of localizing a chromogenic compound (e.g.,liquid-soluble dye) to the distal end-portion (16) of the absorbent wick(6). This specific embodiment allows for the direct visualization ofcapillarity-mediated transfer of the test sample through thechromatography medium (10), thus facilitating monitoring ofdifficult-to-detect samples (e.g., colorless aqueous samples or variousphysiological fluids).

EXAMPLE 5

With reference to FIGS. 1-3, the device is utilized as in Example 1 withthe exception that following the initial disassembly of the assay deviceby the removal of the Sampler component (1) from the Detection component(2), and prior to the actual collection of the test sample, a solution(e.g., buffer) is added to the reagent application port (5). This causesthe saturation of the absorbent wick (6) member and allows for thesolubilizing and capillarity-mediated transfer of a dried or semi-driedsample onto the distal end-portion (16) of the absorbent wick (6).

EXAMPLE 6

With reference to FIGS. 1-3, the device is utilized as in Example 1 withthe exception that after the actual collection of the test sample, asolution with an added reagent such as analyte or analyte analog isadded to the reagent application port (5). This causes the saturation ofthe absorbent wick (6) member and allows for the solubilizing andcapillarity-mediated transfer of a dried or semi-dried sample onto thedistal end-portion (16) of the absorbent wick (6).

EXAMPLE 7

With reference to FIGS. 1-4, the device is utilized in a manner so as toprovide a competitive immunoassay. The competitive assay may behomogeneous or heterogeneous in nature. In a homogeneous assay, all ofthe reactants participating in the competition reaction are mixedtogether and the concentration of the analyte is determined by itseffect on the extent of binding between the receptor and labeled analyteanalog. The observed signal is a direct function of this binding and canbe related to the overall concentration of analyte present in the testsample.

In a heterogeneous competitive binding-pair assay the bound, labeledanalyte or receptor is separate from the free, labeled analyte orreceptor. The concentration of the bound or free fraction issubsequently measured.

In this example, the analyte analog may be added to the device by directapplication to the reagent application port (5) using an externaldelivery device, or, alternately, it may be contained within the reagentcontainers (17) which, when ruptured, deliver the analyte analog to thedevice. The analyte analog may be delivered to the device either beforeor after the actual collection of the test sample. Alternatively, it maybe contained in the wick or tubing (6).

EXAMPLE 8

In addition to ligand-based assays, this type of device is also usefulfor non-ligand assay methodologies such as to quantify blood glucose indiabetes. With reference to FIGS. 1-3, the device is utilized as inExample 4. Test sample potentially containing β-D glucose is assayed,and glucose oxidase is the solution added to the reagent applicationport (5). Glucose oxidase reacts with β-D glucose in the presence ofoxygen to produce D-glucose acid and H₂O₂. Subsequently, horseradishperoxidase (HRPO), 1,7-dihydroxynaphthalene, and 4-aminoantipyrinehydrochloride are added to the distal end-portion (16) of the absorbentwick (6) as chromogenic compounds. Alternatively, the compounds can beadded before or during the assay through the detection port (19) to havethe HRPO system present on the label transfer pad (11), or can belocated directly on (a) the label transfer pad (11), (b) thechromatography medium (10), or (c) the capture zone (13).

EXAMPLE 9

This device can also be used to puncture skin to draw blood or otherfluids for the assay to be run in the device. With reference to FIGS. 6Aand 6B, the retractable sharp (23) is extended. used to perforate theskin, and is then either retracted or broken off and disposed of. Thesample is then collected and assayed as described in the previousexamples.

EXAMPLE 10

In another embodiment of the device, as illustrated in FIG. 7, the sharp(23), which is concave or a hollow tube, is used to perforate the skin.The sample then flows through the sharp to the wick or tubing (6). Thesample is then assayed as described in the previous examples.

While embodiments and applications of the present invention have beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it would be apparent to those individualsskilled within the relevant art that many additional modifications wouldbe possible without departing from the inventive concepts containedherein.

1. A device for performing a binding pair assay comprising: (a) ahousing comprising a sampler member and a detection member, wherein thedevice is capable of being separated into at least two parts comprisingthe sampler member and the detection member, and the device is furthercapable of being assembled into one part; (b) a chromatography regionassociated with the detection member comprising a chromatography mediumhaving a transit zone and a capture zone; (c) a sample collectorassociated with the sampler member that is exposed to receive sampleupon separation of the device into the sampler member and the detectionmember, and wherein the sample collector is in capillary communicatingcontact with the capture zone through the transit zone when the deviceis assembled and; (d) a reagent delivery system positioned relative tothe sampler member for delivering a liquid reagent to the samplecollector, wherein the reagent delivery system is positioned so that theliquid regent is added to the reagent delivery system after the samplermember and detection member are assembled into one part and flowsthrough the sample collector and into the chromatography region.
 2. Thedevice of claim 1 wherein the sample collector comprises an absorbentwick or tubing.
 3. The device of claim 2 wherein the absorbent wickcomprises a bibulous material.
 4. The device of claim 2 wherein thebibulous material is selected from the group consisting of filter paper,chromatographic paper, nitrocellulose, cellulose acetate,polyacrylamide, cross-linked dextrose, and agarose.
 5. The device ofclaim 2 wherein the absorbent wick or tubing comprises at least onevolume indicator near the distal end to indicate sample volume.
 6. Thedevice of claim 2 wherein the sample collector is located in the samplermember.
 7. The device of claim 1 wherein the reagent delivery systemcomprises an absorbent reagent pad.
 8. The device of claim 7 wherein thereagent delivery system is positioned upstream of the sample collector.9. The device of claim 7 wherein the sample collector comprises anabsorbent wick or tubing and conducts liquid reagent from the reagentdelivery system to the transit zone when the device is assembled. 10.The device of claim 7 wherein the reagent delivery system comprises abreakable reagent container that delivers reagent to the end of thesample collector that is in contact with the reagent delivery system,when the container is broken.
 11. The device of claim 10 wherein thereagent delivery system comprises a reagent application port positionedto allow introduction of liquid reagent at the distal end of the samplecollector.
 12. The device of claim 7, wherein the sampler member furthercomprises a sharp for perforating skin to draw blood or other fluids tobe used as the sample.
 13. The device of claim 12 wherein the sharp ispositioned to allow sample to flow along the sharp to the absorbent wickor tubing.
 14. The device of claim 7 wherein the transit zone comprisesa bibulous material and further comprises a label transfer pad.
 15. Thedevice of claim 14 wherein the label transfer pad is located at the endof the transit zone that is opposite of the capture zone.
 16. The deviceof claim 14 wherein the label transfer pad contains labeled moietiesselected from (a) a labeled specific analyte-binding reagent, (b) alabeled analyte analog, (c) components and means for producing withinthe label transfer pad a labeled specific analyte-binding reagent, or(d) components and means for producing within the label transfer pad alabeled specific analyte analog.
 17. The device of claim 16 wherein themeans for producing a labeled specific analyte-binding reagent or alabeled specific analyte analog include a detection port adjacent to thelabel transfer pad positioned to allow the addition of elements requiredto assemble the labeled specific analyte-binding reagent or labeledspecific analyte analog.
 18. The device of claim 17 wherein the samplecollector is positioned to allow capillary communicating contact withthe label transfer pad when the housing is assembled.
 19. The device ofclaim 18 wherein the labeled moieties are distributed on the labeltransfer pad so that the labeled specific analyte-binding reagent orlabeled specific analyte analog becomes mobile upon fluid contactbetween the sample collector and the label transfer pad.
 20. The deviceof claim 18 wherein the labeled moieties are labeled with one or morelabels selected from the group consisting of a radioisotope, aparticulate metal, a dye, one or more components of a catalyzed orenzymatic reaction, and a chemiluminescent compound.
 21. The device ofclaim 7 wherein the simple collector comprises labeled moieties selectedfrom (a) a labeled specific analyte-binding reagent, and (b) a labeledanalyte analog.
 22. The device of claim 21 wherein the labeled moietiesare present at or near the end of the sample collector that is oppositeof the end of the sample collector that is in capillary communicatingcontact with the capture zone through the transit zone when the deviceis assembled.
 23. The device of claim 21 wherein the labeled moietiesare present at or near the end of the sample collector that is incapillary communicating contact with the capture zone through thetransit zone when the device is assembled.
 24. The device of claim 22wherein the labeled moieties are labeled with one or more labelsselected from the group consisting of a radioisotope, a particulatemetal, a dye, one or more components of a catalyzed or enzymaticreaction, and a chemiluminescent compound.
 25. The device of claim 7wherein the reagent delivery system is comprised of a reagentapplication port.
 26. The device of claim 25 wherein the reagentapplication port is positioned to allow introduction of liquid reagentat the end of the sample collector that is in capillary communicatingcontact with the capture zone through the transit zone when the deviceis assembled.
 27. The device of claim 26 wherein the liquid reagentcontains a substance selected from the group consisting of an analyte,an analyte analog, a specific analyte-binding reagent, asignal-generating reagent and an ancillary reagent.
 28. The device ofclaim 7 wherein the reagent delivery system comprises an absorbentreagent pad.
 29. The device of claim 28 wherein the reagent deliverysystem further comprises at least one breakable reagent container andthe absorbent reagent pad is positioned between at least one breakablereagent container and the sample collector.
 30. The device of claim 29wherein the absorbent reagent pad is in capillary communicating contactwith the sample collector.
 31. The device of claim 7 further comprisinga detection port positioned adjacent to the end of the chromatographyregion that makes contact with the sampler member when the device isassembled and shaped to allow addition of reagent to the chromatographyregion.
 32. The device of claim 31 wherein the detection port ispositioned adjacent to the transit zone.
 33. The device of claim 7further comprising an air gap between the reagent delivery system andthe chromatography region, the air gap positioned so that the samplecollector bridges the air gap and creates capillary communicatingcontact between the reagent delivery system and the chromatographyregion.